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Home , Gravitropism, Phototropism, Tropism

How Can Tell Which Way Is Up? Laboratory Exercises To Introduce Gravitropisyn John

Z. Kiss Sean E. Weise Helen G, Kiss

Many people think of plants as The laboratory exercises described structures. In higher plants, a great deal essentially sessile that do not in this paper involve the higher of evidence suggests that amyloplasts actively respond to their environment. , which has become (starch-filled ), which settle or What could be further from the truth! an important in molecular In fact, plants are capable of a variety of sediment to the new lower cell wall research and is the focus of an following reorientation, function as movements, including the dramatic international plant genome project. nastic responses (such as Venus fly trap statoliths. Thus, the idea to explain Based on the material presented here, a gravity perception in plants has been closure) and the less sensational number of plant labora- . These latter movements are referred to as the "starch statolith tory exercises with Arabidopsis that are hypothesis," and the evidence supporting directed growth responses to some type simple in terms of equipment/materi- of external such as gravity als and procedures can be developed. this hypothesis has been reviewed in a (gravitropism, formerly known as These exercises are robust in that they number of publications (e.g. Sack 1997), geotropism) or (). work well even in the hands of intro- including a very accessible article in This paper describes some interesting ductory students, and they can be Scientific American (Evans et al. 1986). exercises that are derived from recent expanded according to the individual Some of the strongest evidence that work, including research that has led to instructor's needs. This paper describes amyloplasts function as statoliths experiments performed on two Space two exercises that have been per- comes from research with mutants of Shuttle missions in 1997 (Kiss et al. formed by beginning college students, Arabidopsis that lack starch (Kiss et al. 1998). and these exercises can easily be per- 1997). These mutants are starchless The study of tropisms can be a useful formed in biology classes in most high since they are missing one of the way to introduce students to plant school settings. biology in high school and introductory enzymes (phosphoglucomutase or college courses. In our experience, pgm) in the final steps of starch students are fascinated by plant move- synthesis. Several studies have shown ments when they are presented in lec- Background that the starchless mutants are much tures and find laboratory experiences less sensitive to gravity, but these on this topic quite engaging. Laboratory mutants still do respond to gravity (i.e. work on plant tropisms can also be Gravitropism in plants can be they are not agravitropic). The upshot used to introduce important concepts divided into three temporal stages: of this work is that plastids (with starch in science such as hypothesis testing, perception, transduction/transmission, in the case of the normal wild-type, quantitative analysis, and the use of and response (i.e. the differential WT, and without starch in the case of statistics. growth in a plant that leads to the mutant) can function in gravity downward or upward curvature). perception. (It is important to note that These laboratory exercises are focused both the WT and mutant plants have on the early events of plant the same number of plastids, but the gravitropism termed "gravity mutant has starchless plastids.) How- perception." Gravitropism and other ever, when starch is present, there is a John Z. Kiss is Professor of at greater density and an increased Miami University in Oxford, OH 45056; e- tropisms in plants have been exten- mail: [email protected], and has sively studied since late in the last total mass of these statoliths, and the a o experiments y on the Space Shuttle. century, and Charles Darwin and his plants do better in terms of gravity Sean E. Weise was an undergraduate son Francis published a book titled The perception and response. botany major at Miami University. Helen Power of Movements in Plants in 1883. In the two related laboratory exer- G. Kiss is in the Office for the cises that follow, students should be Advancement of Scholarship and Teaching (In fact, the Darwins' early experiments and serves as an adjunct faculty member in phototropism are outlined in several able to demonstrate that while both the in Botany at Miami University. introductory biology textbooks.) WT and mutant respond to gravity, the Gravity perception in plants is WT has a greater response. One hypothesized to be mediated by the exercise focuses on gravitropism in the interaction of dense organelles (called INTRODUCING GRAVITROPISM 59

flower stalks of mature plants, and the to provide a humid atmosphere to experiment, and if the plants are left in other involves a study of gravitropism in stimulate germination. At this point, the favorable conditions, they will continue young seedlings. They can be performed pots are placed under continuous to produce additional stalks that can be independently or together, depending on illumination from 40-watt fluorescent used for other experiments.) the instructor and his or her course. bulbs in a "shop" light fixture. Re- Curvature is measured at each time markably, the starchless mutant and WT interval with a protractor, and the plants are identical in morphology and size if then are returned to the box. Sample data they are maintained together under are shown in Table 1. Students can Exercise #I -Flower Stalks continuous illumination. If plants are observe that WT flower stalks curve from Mature Plants grown on a light/dark cycle, then the faster compared to the mutant flower mutant may be smaller and develop at a stalks, even though the growth rates of Materials Needed slower rate compared to the WT. both strains are similar. The data can be Seeds of normal WT Arabidopsis and a However, we have found that the growth presented in table format or values can starchless mutant are available free of rate, in continuous light, of the WT and be used to draw a graph. Instructors can charge (or in some cases, for a small fee) mutant flower stalks is almost identical; also have the students perform some to educators through the Arabidopsis thus potential differences in growth are simple statistical analyses (such as a Biological Resource Center (ABRC) at not significant when evaluating Mest) to determine if the curvature Ohio State University. Orders may be gravitropism. values are significantly different placed through the Internet An alternate to growing the plants in a between the WT and the mutant flower (http://aims.cps.msu.edu/aims/), by e- soil mixture is to use a synthetic stalks. mail ([email protected]), or by substrate, such as Rockwool cubes or If a light-tight box is not practical to standard mail (ABRC, 309 B & Z Oasis floral foam cubes, purchased from implement, it is possible to do this Building, 1735 Neil Ave., Columbus, a greenhouse or gardening supply store. experiment in general room light since OH 43210; telephone: 614-292-9371; The advantage of this method is that the phototropic response is much weaker fax: 614-292-0603). The starchless there is no potential problem with soil than the gravitropic response in these mutant is pgm-1 (stock no. CS210), and spilling when the plants are reoriented. flower stalks. Another variation is the WT strain to use is Columbia (Col- In any case, soil moisture should be simply to do the experiment with the 21stock number CS907). The ABRC will carefully maintained until the seeds WT only, and this exercise still allows not send many seeds, but you can grow germinate, which should be in about students to appreciate the phenomenon more plants for your own seed stock (i.e. three to five days after sowing. Under in plants and to do some data analysis. plants self-pollinate), and each plant will conditions of low humidity, it may take a The upward curvature of WT flower produce thousands of seeds. Other few more days for germination. Seeds stalks is illustrated in Figure 1. materials needed for this exercise will germinate at room temperature (20 This exercise can be enhanced by include soil, pots, a light bank to 22° C), but temperatures above 25° C demonstrating the differing phenotypes (fluorescent "shop" work well), a should be avoided. Following of the WT and starchless mutant. Leaves light-tight box (e.g. photocopy paper germination, plants should be watered as from the plants are excised and left boxes or large shoe boxes), and pro- needed, but too much watering may lead overnight in ethanol to remove the to fungal growth. Arabidopsis is in the chlorophyll. The leaves then are placed mustard family, and the plants will form in an iodine solution (i.e. 2.0 g potass- Preparation rosettes of leaves. Once the plants are at ium iodide and 0.2 g iodine are added to You will need to grow plants in pots the rosette stage, they may need to be 100 mL of ), and the WT will stain until they start forming flower stalks. thinned to avoid overcrowding in the intensely black while there will be no There are many ways to do this, but we pots. About three to four weeks after reaction in the starchless mutant. will provide highlights of one general sowing of seeds, the rosettes will start to Exercise #2-Young method here, and the reader is referred form flower stalks or inflorescences. The Seedlings to the above ABRC site to learn more gravitropism experiments work best on details (if needed) and alternatives in flower stalks that are 2 to 6 cm in length. Materials Needed growing these plants. The good news is Seeds of normal WT Arabidopsis and that Arabidopsis can be grown in a variety a starchless mutant are available through of containers and in many ways, Procedures the ABRC as indicated above. Other including in growth chambers, materials needed include: agar, greenhouses, on window ledges, and After the above preparation, the disposable petri dishes (9 cm diameter), outdoors (after all, it is a real weed). experiment itself is quite simple. Both Parafilm", commercial bleach, The first thing to realize is that Arabi- WT and mutant plants are reoriented 90 disposable Pasteur pipets, small vials, dopsis seeds are very small (0.4 mm in degrees in the dark, and this beginning fluorescent "shop" lights, aluminum foil, diameter), and one has to be careful in of the experiment is referred to as time and protractors. handling them. About one to two dozen 0. We use photocopy paper boxes for the seeds are sown by sprinkling them in dark environment, and reoriented plants Preparation four-inch plastic pots in a commercial are carefully removed from the boxes at An agar mixture, 1.0 to 1.2% (w/v), potting soil such as Metromix 350 or several time intervals (e.g. 0.5 hour, 1.0 is autoclaved and prepared by standard Peter's potting soil. Seeds should not be hour, 2.0 hours, 3.0 hours). Do not microbiological methods. At the same covered with additional soil because worry about the time the plants are time, some water is autoclaved (or they require light for germination. exposed to light since the phototropic sterile water can be purchased). The Several pots can be placed in larger response is relatively weak. (Instructors agar solution is poured into petri dishes, plastic containers that can be covered can excise flower stalks at the end of the which are filled to about one third of with a plastic wrap in order their height. The petri dishes

Table 1. Mean upward curvature in degrees (± standard error) of Arabidopsis flower stalks following a 90-degree reorientation in the dark. Curvature in the normal wild-type is compared to that of a starchless mutant (97 < n < 100).

then are left to cool until the agar solidifies. Prepared agar plates or ready-topour agar that only needs to be heated in a microwave oven can be purchased (e.g. Wards Natural Science Inc., Box 92912, Rochester, NY 14692-9012) if an autoclave is not available. A nutrient agar can also be used and is preferable; recipes can be found at the ARBC Web site listed above or in Kiss et al. (1996). Arabidopsis seeds are surface steri- lized in a 30% (v/v) solution of Clorox" for 15 minutes in a vial, and then are rinsed four times (10 minutes for each step) in sterile water. It helps to place a drop of a detergent such as Triton X-100 or Tween (or even dishwashing liquid) into the Clorox solution and rinse water. Seeds are placed onto the surface of the agar with a Pasteur pipet and are placed approximately 1 cm apart. In the petri dishes, two rows of seeds can be accommodated. Petri dishes are sealed with Parafilm "' and placed on edge in some type of rack so that the surface of the agar is vertical, and an arrow is drawn on the dish to indicate the direction of gravity (Figure 2). At this point, people walking into your lab will realize something new (and maybe strange) is happening since petri dishes are typically placed flat on the bench! The dishes are placed under a fluo- rescent "shop" light for 24 hours. Even though this is a "dark-grown" experi- ment, the seeds have a li ~>,ht require- ment for germination. After the 24hour light treatment, the dishes are wrapped Figure 1. Gravitropic curvature in normal wild-type Arabidopsis flower stalks. At time 0 in aluminum foil and placed in a dark (A), plants were reoriented 90 degrees, and photographs were taken at 1.5 hours (B), 3.0 environment such as a shoe box or in a hours (C), and 5.5 hours (D) following reorientation. The gravity vector is toward the drawer. It also helps to make an arrow bottom of the photographs. Upward curvature is rapid, and some flower stalks on the foil as well as the petri dish itself (arrowhead in panel C) "over-shoot" the gravity vector and then "correct" themselves to indicate the direction of gravity. (panel D). Scale bar = 2 cm. Seedlings that are 1.0 to 1.5 cm in length should develop in about three days following the wrapping of the petri dishes with foil. the aluminum foil is unwrapped. Stu- protractor the angles that hypocotyls are dents should easily see that the WT deviated from the vertical gravity seedlings are oriented relative to gravity vector. If the vertical gravity vector is Procedures while the mutant seedlings are dis- defined as 0 degrees, the mean angle for Petri dishes with four-day-old seed- oriented (Figure 3). This deviation can be the WT should be about 0 degrees, and lings are removed from the dark, and quantified by measuring with a the mean angle for the mutant

INTRODUCING GRAVITROPISM 3

Table 2. Mean upward curvature in degrees (± standard error) of the stem-like hypocotyls of Arabidopsis following a 90-degree reorientation in the dark. Curvature in the normal wild-type is compared to that of a starchless mutant ( 94 < n < 251).

Figure 2. Diagram of petri dish setup used in experiments to study gravitrop- ism in young seedlings. The arrow indi- cates the gravity vector. Figure 4. of Arabidopsis seedlings also be used for measurements of cur- stained with iodine solution. Note the vature of the roots. intense, dark reaction (which indicates Students should be able to see that starch) in the cap (arrowhead) of the mutant does respond to the reori- the wild-type (WT) in panel A while entation but at a much slower rate than there is no reaction in the root cap the WT. (Remember, since growth rates (arrowhead) of the starchless mutant of the WT and mutant seedlings are (M) in panel B. Scale bar = 100 [Lm. nearly identical, growth is not a limiting factor for the mutant.) An important Another useful supplement to these point is that the mutant is not laboratories is a two-page cartoon in the agravitropic as might be the initial August 1997 issue of Discover magazine. hypothesis based on the qualitative Plant gravitropism is introduced in a observations of the orientation experi- humorous, engaging (yet accurate) ment (i.e. the mutant is severely manner in a cartoon titled "Starch wars" disoriented). (Gonick 1997), which has been used in Another simple extension of this an educational outreach program for exercise is to stain the seedlings with high school teachers by North Carolina iodine solution (described above) and State University. then observe them with a compound light microscope. The difference between the WT and mutant is quite Figure 3. Orientation of 4-day-old, Conclusions & Perspectives dramatic as is illustrated by the root cap In terms of scientific conclusions, the dark-grown Arabidopsis seedlings in starch in Figure 4, and the difference in important point is that while both the petri dishes. The gravity vector is the shoots of both strains can also be WT and mutant respond to gravity, the toward the bottom of the photographs. examined. response (i.e. curvature) is greater and Note that the wild-type (WT) is more rapid in the WT. Based on their oriented to gravity (A) while the data, students can make arguments for starchless mutant is relatively and against the starch statolith theory disoriented (B). Scale bar = 5 cm. Supplementory Resources The best way to convey the excitement for plant gravity perception. While it is of the study of plant movements is by clear that starch per se is not needed for should be about 60 degrees. Orientation illustrating the process with motion gravitropism, a full complement of of the roots can also be examined. To pictures. To this end, an excellent starch (as in the WT) provides a better extend this exercise, students can Internet resource with time lapse gravitropic response. Thus, the results perform a time course of curvature movies of tropisms and other plant are still consistent with the essence of a study of the hypocotyls as is outlined movements has been developed by Dr. statolith model (i.e. dense particles above in the flower stalk experiment. Roger Hangarter of Indiana University participating in gravity perception). Petri dishes with seedlings are reori- (http: / /sunflower.bio.indiana.edu. / Perhaps the idea should be called the ented 90 degrees in the dark. The rhangart/plantmotion/). These movies "plastid statolith hypothesis" since it response to reorientation is much slower clearly demonstrate the dramatic appears that plastids (with starch in the than is observed in the flower stalks, so nature of various plant movements and case of the WT, and without starch in time points for observation might feature Arabidopsis as well as other the case of the mutant) can function in include 1 hour, 3 hours, 8 hours, and 24 plants. The topics include seed germi- gravity perception. Instructors will find hours. Seedlings are removed from the nation, gravitropism, phototropism, a more detailed discussion of these ideas dark at these time intervals, and "sleep movements," nutation, and in Salisbury (1993) and Sack (1997). curvature of the hypocotyl is measured flower opening. Particularly relevant From the perspective of teaching with a protractor. Sample data for to the laboratory exercises described in this material, we have found that hypocotyl curvature is shown in Table this paper is the fact that gravitropism students in introductory college 2, but the same seedlings can in Arabidopsis flower stalks is part of biology courses enjoy the laboratory the movie collection. exercises outlined

in this article. They like the idea of for each exercise have been discussed for Kiss, J.Z., Guisinger, M.M., Miller, A.J. & performing experiments that are based on the instructor who has the need and time for Stackhouse, K.S. (1997). Reduced gravi- current research and the relationship of the more complex laboratory experiences. in hypocotyls of starch-deficient exercises to the space program. After all, Finally, we believe that these laboratory mutants of Arabidopsis. Plant and Cell space has been and continues to be a useful exercises will stimulate students to think , 38, 518-525. way to promote and encourage interest in the carefully about their results and to develop Kiss, J.Z., Katembe, W.J. & Edelmann, R.E. natural sciences. Students also value the critical thinking skills that will be useful to (1998). Gravitropism and development of opportunity to gather "real" data that can be them throughout their educational careers. wild-type and starch-deficient mutants of used to evaluate a scientific hypothesis. Arabidopsis during spaceflight. Physiologia While we do not have experience with Plantarum, 102, 493-502. these experiments in a high school setting, Acknowledgments Kiss, J.Z., Wright, J.B. & Caspar, T. (1996). they seem to be appropriate and quite We thank Dr. Richard Edelmann for Gravitropism in roots of intermediatestarch feasible for biology classes at the secondary drawing Figure 2 and the National Aero- mutants of Arabidopsis. Physiologia Plantarum, level. The two exercises are very flexible in nautics and Space Administration for finan- 97, 237-244. that they can be performed independently or cial support (Grant NAG 2-1017). Sack, F.D. (1997). Plastids and gravitropic coupled together as part of a larger project. sensing. Planta, 203, S63-S68. Salisbury, They also can be performed with fairly basic F.B. (1993). Gravitropism: Changing ideas. References Horticultural Reviews, 15, 233-278. equipment and entirely on a macroscopic scale if needed. Appropriate add-on portions Evans, M.E., Moore, R. & Hasenstein, K.H. (1986). How roots respond to gravity. Scientific American, 255(12), 112-119. Gonick, L. (1997). Starch wars. Discover, 18, (August 1997), 32-33.